Method of intracellular sustained-release of drug and preparations

ABSTRACT

A substance of interest is contained in nanospheres which are then encapsulated in fusogenic liposomes to prepare transport carriers that allow physiologically active substances, especially those having high molecular weight such as proteins and genes, to be introduced into cells efficiently and which permit the introduced active substance to be released in the cell at controlled rate. The fusogenic liposomes are prepared by conferring the fusogenic capability of Sendai virus to known liposomes.

TECHNICAL FIELD

This invention relates to the technology of introducing drugs, proteins,genes and other physiologically active substances directly into cellsand then allowing the introduced physiologically active substances to bereleased slowly in the cells. More particularly, the invention relatesto a composition containing a physiologically active substance ofinterest which can be introduced directly into a cell and whichthereafter can be released slowly in the cell. The invention alsorelates to a method for allowing the physiologically active substance ofinterest to be directly introduced cells and then released slowly in thecells.

BACKGROUND ART

For successful gene therapy and treatment with novel vaccines, effortsare being made today in order to ensure that biopolymers such as genesand antigenic proteins are introduced into the cytoplasm directly andefficiently. Unlike low-molecular weight drugs, high-molecular weightsubstances such as genes, antigenic proteins and physiologically activeproteins are not only low in membrane permeability, absorption andtissue migration but they also undergo rapid degradation in blood. Ithas been desired to develop a technology by which those polymeric,physiologically active substances can be introduced into the cytoplasmin a desired and efficient manner without causing damage to the cell.

Liposomes can hold a number of substances and are still biocompatible,so they have drawn researchers' attention as carriers for transportingphysiologically active substances. However, with liposomes, theefficiency of introducing the desired substance into a cell is sow lowthat it can hardly be introduced into the cytoplasm. To deal with thisdifficulty, various proposals have been made, including modifying thesurfaces of liposomes with lectins, antibodies, etc. so that they arepositively bound to cell surfaces. In fact, however, liposomes, whethersurface-modified or not, are taken up by cells via endocytosis, so theyare lysed by an enzyme called lysozyme and the proportion in which thesubstance of interest is actually transferred into the cytoplasm is atan extremely low level.

It was reported that in order to overcome this difficulty, fusogenicliposomes having the fusogenic capability of Sendai virus were developedas carriers that could be directly introduced into the cytoplasm via thecell membrane. Such fusogenic liposomes can be prepared by forming acomplex between a liposome and the coat protein of Sendai virus whichmediates fusion to the cell membrane. The prepared fusogenic liposomeshave an almost comparable fusogenicity to Sendai virus and it has beenreported that by encapsulating genes, proteins or other high-molecularweight substances, the substance of interest can be directly introducedinto the cytoplasm with high efficiency but without causing cell injury[“DDS”, Journal of the Japan Society of DDS, Vol. 13, No. 1, January1998, pp. 21-26 and 27-33].

Even if the fusogenic liposomes are used as carriers for physiologicallyactive substances, the release of the physiologically active substancein cells cannot be controlled, so the substance introduced into the cellis released at a time and its activity (toxicity) is not sustained.Take, for example, the case of introducing a gene into the cytoplasm;the gene is decomposed in the cytoplasm and its expression is notsustained. In the case of a protein having pharmacological activity, theactivity is not sustained and it has to be administered by an increasednumber of times in a larger dose. Therefore, if physiologically activesubstances are introduced into cells directly and efficiently and iftheir release is controlled within the cytoplasm, the intendedphysiological activity can be exhibited efficiently in the cell. It isdesired to develop a transport carrier that permits a substance ofinterest to be introduced into cells and which enables slow release ofthe substance in the cell.

Desired is the development of a safe and stable transport carrier thatallows physiologically active substances, in particular high-molecularweight substances such as proteins and genes, to be introduced intocells efficiently and without damaging the cell membrane and which stillcan control and adjust the release profile of the introduced substancewithin the cell.

DISCLOSURE OF THE INVENTION

The present inventors paid particular attention to nanospheres currentlydeveloped as a technique for controlled release of drugs andaccomplished the present invention by combining nanospheres withfusogenic liposomes.

Active efforts have heretofore been made in an attempt to achievecontrolled in vivo release of drugs and this has spawned a number oftechniques including the nanosphere technology. Nanospheres ascontemplated in the invention are vesicles that are typically made ofpolymeric matrices and which encapsulate a large volume of drugs orhigh-molecular weight substances. The rate for the release of thetrapped drug can be controlled by the appropriate choice for parametersincluding the size of the nanospheres, the type of the matrix-formingpolymer, and the degree of crosslinking within or between molecules ofthe polymer. However, given alone, nanospheres are not easily taken upby cells and even if they are, the uptake is by endocytosis and theefficiency of introduction is very low because of lysis by the enzymecalled lysozyme. The present inventors assumed that by combiningfusogenic liposomes with nanospheres encapsulating a physiologicallyactive substance of interest, it would be possible to achieve efficientintroduction of the nanospheres into the cytoplasm while controlling therelease of the encapsulated physiologically active substance within thecytoplasm. Based on this assumption, the inventors continued theirstudies and finally accomplished the present invention.

The present invention is characterized in that nanospheres encapsulatingdrugs, proteins, genes or other physiologically active substances are inturn encapsulated in liposomes to which is conferred the fusogeniccapability of Sendai virus. Thus, the invention provides a method bywhich the physiologically active substances are not only introduced intocells directly and efficiently but are also released slowly in thecytoplasm. The invention also provides a composition to be used toimplement the method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the time profile of the release ofFITC-dextran from the poly(lactic acid) nanospheres prepared in Example2;

FIG. 2 is an agarose gel electrophoretogram showing how encapsulatedplasmid DNA having the T7 promoter sequence remained stable in thegelatin nanospheres prepared in Example 3 as they were reacted with arat liver homogenate;

FIG. 3 is a graph showing the relationship between the degree ofcrosslinking of gelatin and the release of encapsulated plasmid DNA fromthe gelatin nanospheres prepared in Example 3; and

FIG. 4 is a graph showing the time profile of the intracellularexpression of plasmid DNA having a T7 promoter that was encapsulated ingelatin nanospheres which in turn were encapsulated in the fusogenicliposomes of the invention.

MODES FOR CARRYING OUT THE INVENTION

The nanospheres to be used in the invention may be prepared from anyconventionally known materials by any conventionally known methods aslong as they fit the purpose of the invention. See, for example,Leelarasamee N. et al., J. Microencapsul. 5; 147-57.1988; Singh M. etal., Pharm. Res. 12; 1796-1800, 1995; Rutledge L. C. et al., J. Am.Mosq. Dontrol. Assoc. 12; 39-44, 1996; Li J. K. et al., J. Pharm. Sci.86; 891-895, 1997; and Kofler N. et al., Int. Arch. Allergy. Immunol.113; 424-431, 1997. The nanospheres to be used in the invention haveparticle sizes of 10 nm-100 nm, preferably 20 nm-600 nm.

According to the invention, a desired physiologically active substanceis encapsulated in nanospheres which, in turn, are encapsulated infusogenic liposomes to prepare a composition which allows thephysiologically active substance of interest to be introduced into cellsand then released slowly in the cytoplasm. Liposomes can be used withoutany particular limitations as long as they can hold nanospheres and theycan be prepared by conventional methods including reverse phaseevaporation [Szoka, F. et al., Biochim. Biophys. Acta, Vol. 601 559(1980)], injection of ether [Deamer, D. W., Ann. N.Y. Acad. Sci., Vol.308 250 (1978)] or use of a surfactant [Bruner, J. et al., Biochim.Biophys. Acta, Vol. 455 322 (1976)].

Lipids to form liposome structures may be conventional ones includingphospholipids, cholesterols and nitrogen lipids, and phospholipids aregenerally preferred. Exemplary phospholipids include various naturalphopsholipids and their hydrogenation products, as well as syntheticphospholipids. These phospholipids may be used either alone or inadmixture. If desired, cholesterols, stearylamines, alpha-tocopherolsand other known additives for liposome formation may also be addedduring liposome formation.

Structurally, liposomes may be giant unilamellar vesicles (GUVs), largeunilamellar vesicles (LUVs), multilamellar vesicles (MLVs) and smallunilamellar vesicles (SUVs). The particle size of liposomes is varied as≧1000 nm for GUV, 100 nm-1000 nm for LUV, 200-5000 nm for MLV, and ≦100nm for SUV. For the purpose of the invention, the particle size ispreferably about 10 nm-10 μm, more preferably 10-1000 nm.

Suitable lipids or mixed lipids as liposome formers, together withcholesterols as liposome forming additives, are dissolved in organicsolvents such as tetrahydrofuran, chloroform and ethanol; after themixture is put into a suitable vessel, the solvent is distilled off atreduced pressure to give a film of the liposome former on the innersurface of the vessel. In a typical case, a rotary evaporator is used tomake a film of the lipid on the inner surface of a centrifugal tube. Tothe lipid film, a solution of nanospheres is added as an internalaqueous phase and mixed. The resulting liposome solution is reacted witha fusogenesis promoter such as Sendai virus, inactivated Sendai virus ora fusogenesis promoting protein to prepare fusogenic liposomes. Sendaivirus is inherently nonpathogenic to humans but it is preferablyilluminated with uv light so that the viral RNA is fragmented to ensureutmost safety. The fusogenic liposomes of the invention are safeparticles since they differ from the starting liposomes only in thatthey have been conferred the fusogenic capability of Sendai virus. Thefusogenic liposomes of the invention may typically be formed by themethod of Bangham et al. (Bangham A. D., J. Mol. Biol., 13:238-252,1965).

Examples of the physiologically active substance that can beencapsulated in the fusogenic liposomes of the invention include:various drugs that develop physiological actions when they areintroduced into cells; physiologically active proteins such as hormones,lymphokines and enzymes: antigenic proteins that work as vaccines: genessuch as those which are expressed in cells, plasmids, and genes that areinvolved in the expressing of specified genes that induce expression; aswell as various genes and antisenses that are introduced for genetherapy. The technology of the invention is suitably applied tophysiologically active substances of high molecular weight such asproteins and genes but they can also be applied to various low-molecularweight drugs to give preferred results.

Safety is not the only feature of the fusogenic liposomes of theinvention; they are also highly stable and are easy to handle. Accordingto the method of the invention, a desired substance can be efficientlyintroduced into the cytoplasm without causing injury to the functions ofthe cell membrane which is the barrier to the ingress of substances intothe cytoplasm. By the appropriate choice for the characteristics ofnanospheres such as the starting materials, particle size and the degreeof crosslinking, the profile of the release of the introduced activesubstance in the cytoplasm can be controlled and, in particular, itsslow release can be realized to attain the principal object of theinvention. In addition, physiologically active substances can beintroduced into animals by contacting viable cells with the fusogenicliposomes of the invention.

The following reference example and working examples are provided forfurther illustrating the present invention but are in no way to be takenas limiting.

REFERENCE EXAMPLE Materials in Experiment

Reagents, L-α-dimyristoylphosphatidic acid, egg yoke phosphatidylcholine(PC) and chloroform, were purchased from NOF CORP.; cholesterol,sucrose, p-phenylene diamine and calcium ionophore were purchased fromWako Pure Chemical Industries, Ltd.; carboxy-modified 0.02-μm yellowgreen fluorescent fluospheres, carboxy-modified 0.02-nm red fluorescentfluospheres (nanospheres) and calcium green-1 acetoxymethyl derivativewere purchased from Molecular Probes, Inc.; and Eagle's MEM waspurchased from Nissui Pharmaceutical Co., Ltd.

Methods of Experiment

<Preparation and Purification of Liposomes and Fusogenic Liposomes>

Liposomes were prepared by a partial modification of the method ofBangham et al., supra. Mixed lipids (12.72 mg) [PA:PC:Chol=1:4:5 (molarratio)] were suspended in chloroform and a lipid film was prepared onthe inner surface of a centrifugal tube using a rotary evaporator. Tothe lipid film, 300 μL of a nanosphere/BBS (−) solution was added as aninternal aqueous phase and the two phases were mixed by vortexing toprepare liposomes. By shaking at 37° C. for 2 hours, the solution of theliposomes was reacted with Sendai virus to prepare fusogenic liposomes.To purify the fusogenic liposomes, the reaction solution was layeredonto a 6%. 20%. 30%. 40% and 50% sucrose density gradient andcentrifuged at 24000 rpm and 4° C. for 2 h ours (SW28.1, Beckman). Aftercentrifugation, the fusogenic liposome fractions at the 20%-30% and30%-40% sucrose interfaces were recovered and washed with BSS(−) [20000rpm, 4° C.×40 minutes (SW28.1, Beckman)]. Before use in the experiment,the fusogenic liposomes were illuminated with uv light (2000 J/m³) inorder to fragment the RNA of Sendai virus.

<R18 labelling of liposomes and fusogenic liposomes>

A portion (0.1 mM) of R18/ethanol solution was added in a {fraction(1/100)} volume to the liposomes or fusogenic liposomes were conditionedto have the same fluorescence intensity of 150000 (Ex, 490 nm; Em, 515nm) and reaction was performed at room temperature for 1 hour. Theunreacted R18 was removed by centrifuging at 25000 rpm and 4° C. for 40minutes (SW55, Beckman).

<Cultured cells>

Mouse fibroblast cells Ltk⁻were cultured in an Eagle's MEM mediumcontaining 10% fetal calf serum (FCS).

<Introducing Nanospheres Into Cultured Cells>

4×10⁴ Ltk⁻cells were seeded on a 4-well chamber slide. After one day,the cells were washed with PBS(−) and exposed for 1 minute to the actionof the nanospheres, liposomes or fusogenic liposomes (fluorescenceintensity, 100000; Ex, 490 nm; Em, 515 nm). After washing with PBS(−),0.1% p-phenylenediamine/Eagle's MEM medium was added as an anti-stainagent.

<Examination by Confocal Laser Microscopy>

Examination was made with a confocal laser scanning fluorescencemicroscope, MRC-1024 (BIO-RAD). After excitation with a Kr/Ar laser, thenanospheres (yellow green fluorescent) were examined at Ex of 488 nm andEm of 540 nm (540 DF) and R18 was examined at Ex of 568 nm and Em of 585nm (585 LP).

<Measuring the Efficiency of Introduction Into Cells With the Aid ofCa²′ Indicator>

To the nanospheres (red fluorescent), calcium green-1 acetoxymethylderivative was added as a fluorescent Ca²⁺ indicator to give aconcentration of 20 mM and mixed at room temperature for 1 hour so thatit was adsorbed on the nanospheres. The unadsorbed calcium green-1 wasremoved by dialysis (Spectra/Por Membranes MWCO:12-14000) and bufferreplacement was performed to prepare liposomes and fusogenic liposomes.2×10⁵ Ltk⁻cells that had been seeded on 6-well plates one day beforewere washed with PBS(−) and exposed for 1 minute to the action of thecalcium green-1 acetoxymethyl derivative, calcium green-1 adsorbednanospheres, liposomes or fusogenic liposomes which were all conditionedto have the same fluorescence intensity of 2700 (Ex. 488 nm; Em, 534nm). Thereafter, the cells were washed with PBS(−), cultured in anEagle's MEM medium for 1 hour, stripped from the medium with trypsin andsubjected to the measurement of fluorescence intensity (Ex, 488 nm; Em,534 nm). Thereafter, a calcium ionophore/DMSO solution was added to givea concentration of 0.1 mg/mL and fluorescence intensity was measured atEx=488 nm and Em=534 nm.

Results and Discussion

To begin with, the fluorescence labelled nanospheres having a diameterof 20 nm were examined with a confocal laser microscope at amagnification of 10000 in order to see whether they could beencapsulated in the liposomes or fusogenic liposomes. As a result,aggregated images were obtained but the fluorescence of the 20-nm⁴nanospheres was observed only in the positions on transmissionmicrographs where the liposomes or fusogenic liposomes were observed.Thus, it became clear that the nanospheres could be encapsulated intoliposomes and that using such liposomes, one could prepare fusogenicliposomes having the nanospheres encapsulated therein. It was confirmedthat several nanospheres had been encapsulated in each liposome orfusogenic liposome.

The thus prepared fusogenic and non-fusogenic liposomes having thenanospheres encapsulated therein were labelled with R18 and theefficiency of nanosphere introduction into Ltk³¹ cells was determined.Fluorescence was hardly detectable in the cells on which thenon-fusogenic, nanosphere encapsulating liposomes or the nanospheresalone were acted upon; on the other hand, a large number of nanosphereswere introduced into the cells on which the fusogenic liposomes had beenacted. Unlike the nanospheres, the distribution of the red fluorescencefrom R18 used as the marker of the fusogenic liposomes was not observedwithin the cells but observed on the cell membrane. Section images ofthe Ltk³¹ cells on which the fusogenic liposomes had been acted weresuccessively taken in thicknesses of 4 μm as calculated from the surfaceof attachment; nanospheres were clearly visible inside the cell membranein conformity with the cell shape, indicating the presence of R18fluorescence on the cell membrane. Thus, the fusogenic capability of thefusogenic liposomes was reconfirmed and it was suggested that even thenanospheres in suspension could be efficiently introduced into thecytoplasm by using the fusogenic liposomes.

These phenomena were further studied using a calcium green-1acetoxymethyl derivative as a Ca²⁺ indicator. The calcium green-iacetoxymethyl derivative does not emit fluorescence on its own but whenin a cell, it is hydrolyzed by an endogenous esterase to form a chelatewith Ca²⁺, whereupon it emits strong fluorescence. Using this nature ofthe calcium green-1 acetoxymethyl derivative, the present inventorsprepared nanospheres having said derivative adsorbed on the surface. Thenanospheres were then encapsulated into fusogenic liposomes and theefficiency of introduction of the nanospheres into the cytoplasm wasevaluated. In substantial absence of calcium in the cytoplasm,fluorescence was hardly detectable in each of the groups under test.Even when the intracellular calcium concentration was elevated by theaction of calcium ionophore, no fluorescence was detected in the cellson which the nanospheres alone or the nanosphere-encapsulating,non-fusogenic liposomes had been acted. Fluorescence was barelydetectable in the cells on which was acted the calcium green-1acetoxymethyl derivative alone as it was passively transported into thecytoplasm. However, the cells treated with the nanosphere-encapsulating,fusogenic liposomes emitted at least 20 times as much fluorescence asthe cells treated solely with the calcium green-1 acetoxymethylderivative, thus demonstrating the ability of the fusogenic liposomes topermit efficient and positive introduction of nanospheres into thecytoplasm.

These results show that using the fusogenic liposomes,20-nm^(⊕)nanospheres were directly introduced into the cytoplasm with avery high efficiency. The results also show that using the fusogenicliposomes, not only molecules but also vesicles could be introduced intothe cytoplasm. It is therefore shown that the fusogenic liposomes canachieve not only spatial dynamic control of substances within cells butalso temporal dynamic control such as slow release of drugs from thevesicles introduced into the cell.

EXAMPLE 1 Fusogenic Liposome Assisted Introduction of Rhodamine-PEEncapsulating Polyurea Nanospheres and FITC-Dextran EncapsulatingPoly(lactic acid) Nanospheres into the Cytoplasm

It was found in the Reference Example that using the fusogenicliposomes, even the nanospheres or vesicles having a diameter of 20 nmcould be efficiently introduced into the cytoplasm. Based on thisfinding, the present inventors prepared nanospheres capable of slowrelease of drugs, attempted to introduce them into the cytoplasm andchecked for their stability in the cell. Two models were prepared forthe future nanospheres capable of slow release of drugs into thecytoplasm; one was polyurea nanospheres encapsulating rhodamine-PE as afat-soluble drug model and the other was poly(lactic acid) nanospheres(PLA nanospheres) encapsulating FITC-dextran as a model for a drug inaqueous solution.

These two kinds of nanospheres were encapsulated in fusogenic liposomes.After allowing the liposomes to act on cells, their stability in thecell was examined by confocal laser microscopy.

Materials in Experiment

Diacyl phosphatidylethanolamine-N-lissamine rhodamine B sulfonyl(rhodamine-PE) was purchased from Avanti Polar Lipods, INc. Polyvinylalcohol (PVA), tolylene diisocyanate (TDI), soybean oi, poly(lacticacid) (MW=5000; PLA), Span 80 and methylene chloride were purchased fromWako Pure Chemical Industries, Ltd. FITC-Dextran (MW=150000) waspurchased from Sigma. Other reagents were the same or substantially thesame as those described in the Reference Example (see under “Materialsin Experiment”).

Methods of Experiment

<Preparing Rhodamine-PE Encapsulating Polyurea Nanospheres (Ph-PolyureaNanospheres)>

The Ph-polyurea nanospheres were prepared by interfacial polymerizationas follows. A portion (20 mL) of 0.5% PVA/H₂O was homogenized at 20000rpm; a mixture of TDI (2.4 g), soyben oil (2.5 g) and rhodamine-PE (0.5mg/500 mL) was added slowly and homogenized for 3 minutes to prepare anoil-in-water (O/W) emulsion. Another portion (100 mL) of 0.5% PVA/H₂Owas homogenized at 10000 rpm and the previously homogenized mixture wasadded slowly and homogenized for an additional 10 minutes to prepare anoil-in-water-in-water [(O/W)/W] emulsion. To reinforce the surfaces ofthe nanospheres, 100 mL of 0.5% PVA/H₂O was added and homogenized for 20minutes, then homogenized at 7000 rpm for 2 hours to prepare Rh-polyureananospheres. To purify, the polyurea nanospheres were layered onto a10%, 20%, 30%, 40% and 50% sucrose step density gradient and centrifugedat 24000 rpm for 2 hours (SW28.1, Beckman). The Rh-polyurea nanospherefraction at the the 30%-40% sucrose interface was recovered and used inthe experiment.

<Preparing FITC-Dextran Encapsulating PLA Nanospheres (FITC-PLANanospheres)>

The FITC-PLA nanospheres were prepared by interfacial precipitation asfollows. One gram of PLA and 1 mL of Span 80 were added to 30 mL ofCH₃Cl₂ and homogenized at 10000 rpm; 5 mL of FITC/dextran (100 mg/mL)was added slowly and homogenized for 1 minute to prepare a water-in-oil(W/O) emulsion; this emulsion was slowly added to 100 mL of 0.5% PVA/H₂Oand homogenized for an additional 5 minutes to prepare awater-in-oil-in-water [(W/O)/W emulsion. The emulsion was furtherhomogenized at 7000 rpm for 1 hour to prepare FITC-PLA nanospheres whichwere purified by sucrose step density-gradient centrifugation. TheFITC-PLA nanosphere fractions at the 0-10% and 10-20% sucrose interfaceswere recovered and used in the experiment.

<Preparing and Purifying Fusogenic Liposomes>

Liposomes and fusogenic liposomes were prepared by a modification of theprocedures described in the Reference Example (see under “Preparing andpurifying liposomes and fusogenic liposomes” in “Methods ofExperiment”). In the preparation of Rh-polyurea nanosphere encapsulatingfusogenic liposomes, the method of Bangham et al. was repeated, with thefluorescence intensity of the nanospheres being adjusted to 50000 (Ex,550 nm; Em, 590 nm), to prepare liposomes. Thereafter, the liposomesolution was layered onto a 10%, 20%, 30%, 40% and 50% sucrose densitygradient and centrifuged at 24000 rpm for 2 hours (SW28.1, Beckman); thefraction at the 20-30% sucrose interface was recovered and the liposomeswere purified. The purified liposomes were reacted with Sendai virus andthe reaction product was layered onto a 20%. 30%, 35%, 45% and 50%sucrose density gradient and centrifuged at 24000 rpm for 2 hours(SW28.1, Beckman): the fractions at the 30-35% and 35%-40% sucroseinterfaces were recovered to give a purified form of Rh-polyureananosphere encapsulating fusogenic liposomes.

In the preparation of FITC-PLA nanosphere encapsulating fusogenicliposomes, liposomes were first prepared, with the fluorescenceintensity of the nanospheres being adjusted to 10000 (Ex, 490 nm; Em,529 nm). The liposomes were reacted with Sendai virus and the reactionproduct was layered onto a 10%, 20%, 30%, 40% and 50% sucrose densitygradient and centrifuged at 24000 rpm for 2 hours (SW28.1, Beckman). Thefusogenic liposomes prepared from the nanospheres recovered from thefractions at the 0-10% and 10-20% sucrose interfaces were present at the10-20% and 20%-30% sucrose interfaces, respectively; these fractionswere recovered to give a purified form of FITC-PLA nanosphereencapsulating fusogenic liposomes.

<Introducing Nanospheres Into Cultured Cells>

4×10⁴ Ltk⁻ cells were seeded on 4-well chamber slides. After one day,the cells were washed with PBS(−) and exposed for 1 hour to the actionof the Rh-polyurea nanospheres, Rh-polyurea nanosphere encapsulatingfusogenic liposomes (fluorescence intensity, 10000; Ex, 550 nm; Em. 590nm), FITC-PLA nanospheres, and FITC-PLA nanosphere encapsulatingfusogenic liposomes (fluorescence intensity, 700: Ex, 490 nm: Em, 520nm). After washing with PBS(−), the cells were cultured in an Eagle'sMEM medium. After one day, the medium was replaced by an anti-stainagent, or a 0.1% p-phenylenediamine/Eagle's MEM medium and the cellswere examined.

<Examination by Confocal Laser Microscopy>

Examination was made in accordance with the procedure described in theReference Example (see under “Examination by confocal laser microscopy”in “Methods of Experiment”). For rhodamine-PE, the conditions were Ex of568 nm and Em of 585 nm (585 LP); for FITC-dextran, the conditions wereEx of 488 nm and Em of 540 nm (540 DF). The prepared Rh-polyureananospheres and FITC-PLA nanospheres, as well as the fusogenic liposomesencapsulating the RH-polyurea nanospheres and the fusogenic liposomesencapsulating the FITC-PLA nanospheres were examined by confocal lasermicroscopy; without doubt, the two kinds of nanospheres were prepared.

The fluorescence of the substances encapsulated in the two kinds ofnanospheres occurred in the positions on transmission micrographs wherethe fusogenic liposomes were observed. Thus, it became clear that thepolyurea nanospheres and PLA nanospheres could be encapsulated intofusogenic liposomes. According to the transmission photographs, thepolyurea nanospheres prepared in Example 1 had particle sizes of about200 nm and the PLA nanospheres had particle sizes of about 500 nm. Thesenanospheres were encapsulated in the fusogenic liposomes, indicatingthat even fairly large nanospheres could be encapsulated in thefusogenic liposomes. Since the polyurea nanospheres could beencapsulated in the fusogenic liposomes, it became clear that accordingto the invention, one could produce fusogenic liposomes thatencapsulated a large volume of hydrophobic drugs which had beenencapsulated with only low efficiency by the conventional methods ofliposome preparation. An examination made one day after the nanosphereencapsulating fusogenic liposomes were allowed to act on the Ltk⁻ cellsoffered positive evidence for the presence of the nanospheres within thecells. The Ltk⁻ cells into which the nanospheres were introduced bymeans of the fusogenic liposomes showed no morphological changes afterthe lapse of one day and this indicates the absence of superficial cellinjury.

These results show that the fusogenic liposomes can introduce 100-500 nmnanospheres into the cytoplasm and that the introduced nanospheresremain stable in the cytoplasm even after the passage of a day. Based onthis finding, the inventors studied the release of drugs from thenanospheres in Example 2.

EXAMPLE 2 Slow Drug Release from FITC-Dextran Encapsulating Poly(lacticacid) Nanospheres

It was found in Example 1 that the prepared nanospheres could beintroduced into the cytoplasm by means of the fusogenic liposomes andthat they remained stable within the cell for at least 24 hours.However, if these nanospheres are incapable of releasing drugs at slowrate, it is of course impossible to have drugs released slowly in cells.The present inventors therefore chose FITC-dextran as a model drug andconducted an experiment to evaluate its slow releasability from theprepared FITC-PLA nanospheres.

Materials in Experiment

The reagents were the same or substantially the same as those describedin Example 1 (see under “Materials in Experiment”).

Methods of Experiment

<Evaluating the Slow Release of Drug From FITC-Dextran Encapsulating PLANanospheres>

The FITC-PLA nanospheres were suspended in BSS(−) at a fluorescenceintensity of 100000 (Ex, 490 nm; Em, 520 nm) and incubated at 37° C. Atdays 0, 1, 2, 3 and 4, centrifuge was conducted at 10000 rpm to removethe nanospheres and the amount of FITC-dextran in the supernatant wasmeasured with a fluorescence photometer at Ex of 490 nm and Em of 520nm.

Results and Discussion

The release of FITC-dextran from the FITC-PLA nanospheres prepared inExample 1 was observed. As FIG. 1 shows, the FITC-dextran was releasedin only about 6% even at day 4 and its slow release was evident. The PLAnanospheres or microspheres are generally said to release drugs over aperiod from about 3 weeks to three months and in one report, the releasewas ten-odd percent at day 3. Compared to the data reported previously,the PLA nanospheres prepared in Example 1 allow for somewhat slower drugrelease. However, the rate of drug release from the PLA nanospheres canbe controlled by adjusting the molecular weight of PLA and the size ofspheres, so the slow release characteristics of drugs in the cytoplasmcan be controlled by appropriately changing the conditions of nanospherepreparation.

Considering the result of Example 2 in combination with that of Example1, it is concluded that the nanospheres allowed the drug to be slowlyreleased in the cytoplasm.

EXAMPLE 3 Studies on the Stabilization and Controlled Release of PlasmidDNA Encapsulated in Gelatin Nanospheres

The T7 expression system enables efficient gene expression ifbacteriophage T7 RNA polymerase and plasmid DNA having the T7 promotersequence are both present in the cytoplasm. However, due to the lowstability of plasmid DNA in the cytoplasm, gene is rapidly decomposedaway, leading to only a very brief period of gene expression. Therefore,in order for the T7 expression system to express genes intherapeutically necessary amount for the necessary period, the genes inthe cytoplasm must be controlled dynamically by stabilizing the plasmidDNA in the cytoplasm and allowing it to be released slowly. Nanospheresare used as formulations capable of slow release of drugs and as shownin the Reference Examples and Examples 1 and 2, they can not only beintroduced into but can also be released slowly in the cytoplasm. Whilenanospheres can be made of various materials including PLA mentioned inExample 1, gelatin has particularly good biocompatibility and can easilyform gelatin nanospheres by coacervation with plasmid DNA and otherhigh-molecular weight substances. For experimental purposes, gelatinnanospheres are considered to provide ease in evaluating the slowintracellular release of drugs using gene expression as a marker becausethe release pattern can be conveniently controlled by crosslinking andbecause the gelatin nanospheres permit faster drug release than the PLAnanospheres which are generally held to be capable of fast enough drugrelease. With these points taken into consideration, the gelatinnanospheres are believed to be most suitable for use at the first stageof applying the slow intracellular release to the T7 expression system.Therefore, in Example 3, gelatin nanospheres containing plasmid DNA wereprepared by phase separation and studies were made on their ability tostabilize plasmid DNA, as well as the control of its release.

Materials in Experiment

Gelatin (type A, produced from porcine skin) was purchased from Sigma.Other reagents, 2-morpholinoethanesulfonic acid monohydrate (MES),1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC, WSC),Na₂SO₄ and glycine were purchased from Wako Pure Chemical Industries,Ltd. Pentobarbital was purchased from Dainippon Pharmaceutical Co., Ltd.The other reagents were the same or substantially the same as thosedescribed in the Reference Example (see under “Materials inExperiment”).

Methods of Experiment

<Preparing pT7-β-Globin-IRES-L-polyA Encapsulating Gelatin Nanospheres>

The pT7-β-globin-IRES-L-polyA encapsulating gelatin nanospheres wereprepared by phase separation. A hundred milliliters of 5% gelatinsolution and a 45 mM Na₂SO₄ solution containingpT7-β-globin-IRES-L-polyA(0.2 mg/mL) were mixed in a 1.5 mL Eppendorftube and incubated in a water bath at 57° C. for 10 minutes. Thereafter,the mixture was vortexed for 1 minute, layered on top of a 35%, 55% and66% sucrose density gradient and centrifuged at 19000 rpm and 20° C. for1 hour (SW55, Beckman). A 55% sucrose layer containing thepT7-β-globin-IRES-L-polyA encapsulating gelatin nanospheres wasrecovered and subjected to the experiment.

<Crosslinking the Gelatin Nanospheres>

After preparing the gelatin nanospheres, the 55% sucrose layer wasdiluted 2-fold and reacted with an MES buffer solution (0.2 M MES, 0.1or 0.5 mg/mL of EDC, pH 4.5) that was added in a {fraction (1/10)}volume. Thirty minutes later, glycine was added to give 0.2 M, therebyquenching the reaction.

<Preparing Fraction S-9>

Wistar rats were fasted overnight, anesthetized with peritoneallyadministered pentobarbital and had the blood taken from the heart untildeath. Ice-cooled 0.15 M KCl was perfused through the portal vein intothe liver in order to remove a maximum volume of the hepatic blood. Theliver was extracted and washed with ice-cooled 0.15 M KCl; after addingice-cooled 0.15 M KCl in an amount three times its weight, the liver washomogenized under cooling with ice. The homogenate was centrifuged at9000 g and 4° C. for 10 minutes. The supernatant was used as fractionS-9. This fraction was quick frozen with liquid nitrogen and stored at−80° C.

<Studying the Stabilization of Plasmid DNA>

The pT7-β-globin-IRES-L-polyA encapsulating gelatin nanospheres were putinto a 5% solution of fraction S-9 and reaction was performed at 37° C.After a specified time, the S-9 solution was removed by centrifuging andthe remaining nanospheres were destroyed by treatment with trypsin; theplasmid DNA was stained with ethidium bromide and its stability wasevaluated by agarose gel electrophoresis.

<Release of Plasmid DNA From Gelatin Nanospheres>

The pT7-β-globin-IRES-L-polyA encapsulating gelatin nanospheres werecrosslinked at various concentrations by the method described above andtreated in 1.25 mg/mL of trypsin for a specified time. Thereafter, thenanospheres were removed by centrifuging and the amount of plasmid DNAreleased was determined with DABA.

Results and Discussion

In order to evaluate the stability of plasmid DNA in cells, thedegradability of plasmid DNA was examined by agarose gel electrophoresisusing fraction S-9 of the rat liver homogenate (FIG. 2). When the nakedplasmid DNA was reacted with fraction S-9, it was substantially degradedwithin 30 minutes; on the other hand, when it was encapsulated withinthe gelatin nanospheres, the plasmid DNA remained stable at 60 minutesand thereafter. This showed that encapsulating plasmid DNA withinnanospheres was effective in increasing its stability. According to therelease pattern of plasmid DNA from the gelatin nanospheres in trypsinsolution after treatment with various concentrations of the crosslinker,the release of plasmid DNA was suppressed by increasing theconcentration of the crosslinker (FIG. 3).

In the T7 expression system which expresses genes in the cytoplasm whereplasmid DNA tends to degrade faster than in the nucleus, the stabilityof the plasmid DNA in nanospheres and the rate of its release from themare two key factors in adjusting the amount and period of geneexpression. The results of Example 3 suggest that the stability ofplasmid DNA is improved by encapsulating them within gelatin nanospheresand that the rate of release of a drug (which is plasmid DNA in theexperiment described above) can be controlled by altering the conditionsof nanosphere preparation. This is evidence for the possibility ofadjusting the amount and period of gene expression.

EXAMPLE 4 Gene Expression by Plasmid DNA Encapsulated in GelatinNanospheres Encapsulated in Fusogenic Liposomes

In Example 3, it was shown that the intracellular stability of plasmidDNA was improved by encapsulating it in nanospheres and that the amountof release of plasmid DNA could be controlled by treating gelatinnanospheres with a crosslinker. Based on these results, the presentinventors attempted to increase the period and amount of gene expressionby the T7 expression system. To this end, in Example 4, gelatinnanospheres encapsulating PT7-β-globin-IRES-L-polyA which was aluciferase expressing plasmid DNA having the T7 promoter wereencapsulated in fusogenic liposomes and introduced into T7 RNApolymerase producing cells to see how the plasmid DNA slowly released inthe cell would affect the pattern of gene expression by the T7expression system.

Materials in Experiment

As a luciferase assay system, PicaGene, a product of TOYO INK MFG. CO.,LTD. was used. The other reagents were the same or substantially thesame as those described in the Reference Example and Examples 1-3 (seeunder “Materials in Experiment”).

Methods of Experiment

<Preparing and purifying fusogenic liposomes>

Fusogenic liposomes were prepared by a partial modification of theprocedures described in the Reference Example (see under “Preparing andpurifying liposomes and fusogenic liposomes”). To be specific, Sendaivirus whose RNA was fragmented by illumination with uv light (2000 J/m²)were reacted with liposomes to obtain fusogenic liposomes.

<Cultured Cells>

Monkey kidney epithelial cells LLC-MK2#10 were cultured in an Eagle'sMEM medium containing 10% fetal calf serum (FCS).

<Introducing a Substance Into Cultured Cells>

5×10⁴ LLC-MK2#10 cells were seeded on 12-well plates. After one day, thecells were washed with PBS(−). In a separate step,pT7-β-globin-IRES-L-polyA encapsulating fusogenic liposomes,pT7-β-globin-IRES-L-polyA encapsulating gelatin nanosphere encapsulatingfusogenic liposomes and pT7-β-globin-IRES-L-polyA encapsulating gelatinnanospheres were diluted in a serum-free Eagle's MEM medium at anappropriate concentration to incorporate the same amount of genes. Suchfusogenic liposomes or nanospheres were allowed to act on the washedcells for 1 hour. After washing with PBS(−), the cells were cultured inan Eagle's MEM medium.

<Measuring the Luciferase Activity>

Luciferase activity was measured with the luciferase assay system and aluminometer (Lumit LB 9507, Berthold). The activity was expressed interms of relative light unit/well.

For other methods of experiment, see “Methods of Experiment” in Example3.

Results and Discussion

The plasmid DNA containing nanospheres crosslinked with 0.01 mg/mL ofEDC were encapsulated in the fusogenic liposomes, introduced into T7 RNApolymerase producing cells and checked for their effectiveness using thedaily profile of gene expression as a marker (FIG. 4). The fusogenicliposomes encapsulating only plasmid DNA and the plasmid DNAencapsulating nanospheres, both having been processed to have the sameamount of genes, were similarly allowed to act on the cells and thedaily profile of luciferase activity was measured. The result wassubstantially nil. In the group treated with the fusogenic liposomesencapsulating the plasmid DNA encapsulating nanospheres, very highluciferase activity was observed at day 1 and day 2 after geneintroduction. The efficiency of gene expression was substantially thesame at day 1 and day 2 after gene introduction. The pattern of geneexpression in the T7 expression system has been shown to be such thatgene expression disappears significantly at day 1 of gene introduction,so the result of Example 4 seems to suggest that a certain extension ofthe period of gene expression could be achieved by incorporating a genein nanospheres and introducing it into the cytoplasm. Under theconditions used in Example 4, the gene expression dropped at day 4.However, in Example 4, the efficiency of gene expression increased andthe expressed gene remained stable for at least 2 days. Hopefully, theamount and period of gene expression can be adjusted by altering theconditions of nanosphere preparation.

1. A slow-release composition for introducing a physiologically activesubstance into the cytoplasm, comprising nanospheres that encapsulatethe physiologically active substance and which are encapsulated inliposomes having fusogenic capability conferred by reaction with aSendai virus.
 2. The composition according to claim 1, wherein thenanospheres have a particle size of 10-600 nm.
 3. A slow-releasecomposition for introducing a physiologically active substance into thecytoplasm, comprising nanospheres that encapsulate the physiologicallyactive substance and which are encapsulated in liposomes havingfusogenic capability conferred by reaction with a Sendai virus, whereinthe physiologically active substance is selected from the groupconsisting of low-molecular weight drugs, proteins and genes.
 4. Aprocess for producing a composition that contains a physiologicallyactive substance and which allows it to be slowly released in thecytoplasm, comprising the steps of encapsulating a physiologicallyactive substance in nanospheres and encapsulating said nanospheres inliposomes having fusogenic capability conferred by reaction with aSendai virus.
 5. A method of introducing a physiologically activesubstance into an animal by contacting a viable cell with thecomposition according to claim
 1. 6. The composition according to claim2, wherein the physiologically active substance is selected from thegroup consisting of low-molecular weight drugs, proteins and genes.
 7. Amethod of introducing a physiologically active substance into an animalby contacting a viable cell with the composition according to claim 2.8. A method of introducing a physiologically active substance into ananimal by contacting a viable cell with the position according to claim3.
 9. A method of introducing a physiologically active substance into ananimal by contacting a viable cell with the composition according toclaim 6.